The present invention provides methods for the diagnosis and prognosis of cancer, particularly metastatic cancer.
Cancer, its development and treatment is a major health concern. The standard treatments available are few and directed to specific types of cancer, and provide no absolute guarantee of success. Most treatments rely on an approach that involves killing off rapidly growing cells in the hope that rapidly growing cancerous cells will succumb, either to the treatment, or at least be sufficiently reduced in numbers to allow the body""s system to eliminate the remainder. However most, of these treatments are non-specific to cancer cells and adversely effect non-malignant cells. Many cancers although having some phenotype relationship are quite diverse. Yet, what treatment works most effectively for one cancer may not be the best means for treating another cancer. Consequently, an appreciation of the severity of the condition must be made before beginning many therapies. In order to most effective, these treatments require not only an early detection of the malignancy, but an appreciation of the severity of the malignancy. Currently, it can be difficult to distinguish cells at a molecular level as it relates to effect on treatment. Thus, methods of being able to screen malignant cells and better understand their disease state are desirable.
While different forms of cancer have different properties, one factor which many cancers share is that they can metastasize. Until such time as metastasis occurs, a tumor, although it may be malignant, is confined to one area of the body. This may cause discomfort and/or pain, or even lead to more serious problems including death, but if it can be located, it may be surgically removed and, if done with adequate care, be treatable. However, once metastasis sets in, cancerous cells have invaded the body and while surgical resection may remove the parent tumor, this does not address other tumors. Only chemotherapy, or some particular form of targeting therapy, then stands any chance of success.
The process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location (Fidler, et al., Adv. Cancer Res. 28, 149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238 (1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and Zetter, V. Eng. J. Med. 322, 605-612 (1990)). Success in establishing metastatic deposits requires tumor cells to be able to accomplish these steps sequentially. Common to many steps of the metastatic process is a requirement for motility. The enhanced movement of malignant tumor cells is a major contributor to the progression of the disease toward metastasis. Increased cell motility has been associated with enhanced metastatic potential in animal as well as human tumors (Hosaka, et al., Gann 69, 273-276 (1978) and Haemmerlin, et al., Int. J. Cancer 27, 603-610 (1981)).
Identifying factors that are associated with onset of tumor metastasis is extremely important. In addition, to using such factors for diagnosis and prognosis, those factors are an important site for identifying new compounds that can be used for treatment and as a target for treatment identifying new modes of treatment such as inhibition of metastasis is highly desirable.
Tumor angiogenesis is essential for both primary tumor expansion and metastatic tumor spread, and angiogenesis itself requires ECM degradation (Blood et al., Biochim. Biophys. Acta 1032:89-118 (1990)). Thus, malignancy is a systemic disease in which interactions between the neoplastic cells and their environment play a crucial role during evolution of the pathological process (Fidler, I. J. Cancer Metastasis Rev. 5:29-49 (1986)).
There is mounting evidence that VEGF may be a major regulator of angiogenesis (reviewed in Ferrara, et al., Endocr. Rev., 13, 18-32 (1992); Klagsbrun, et al., Curr. Biol., 3, 699-702 (1993); Ferrara, et al., Biochemi. Biophjs. Res. Commun., 161, 851-858 (1989) ). VEGF was initially purified from the conditioned media of folliculostellate cells (Ferrara, et al., Biochem. Biophjs. Res. Common., 161, 851-858 (1989)) and from a variety of tumor cell lines (Myoken, et al., Proc. Natl. Acad. Sci. USA, 88:5819-5823 (1991); Plouet. et al., EMBO. J., 8:3801-3806 (1991)). VEGF was found to be identical to vascular permeability factor, a regulator of blood vessel permeability that was purified from the conditioned medium of U937 cells at the same time (Keck, et al., Science, 246:1309-1312 (1989)). VEGF is a specific mitogen for endothelial cells (EC) in vitro and a potent angiogenic factor in vivo. The expression of VEGF is up-regulated in tissue undergoing vascularization during embryogenesis and the female reproductive cycle (Brier, et al., Development, 114:521-532 (1992); Shweiki, et al., J. Clin. Invest., 91:2235-2243 (1993)). High levels of VEGF are expressed in various types of tumors, but not in normal tissue, in response to tumor-induced hypoxia (Shweiki, et al., Nature 359:843-846 (1992); Dvorak et al., J. Exp. Med., 174:1275-1278 (1991); Plate, et al., Cancer Res., 53:5822-5827; Ikea, et al., J. Biol. Chem., 270:19761-19766 (1986)). Treatment of tumors with monoclonal antibodies directed against VEGF resulted in a dramatic reduction in tumor mass due to the suppression of tumor angiogeneis (Kim, et al., Nature, 382:841-844 (1993)). VEGF appears to play a principle role in many pathological states and processes related to neovascularization. Regulation of VEGF expression in affected tissues could therefore be key in treatment or prevention of VEGF induced neovascularization/angiogenesis.
VEGF exists in a number of different isoforms that are produced by alternative splicing from a single gene containing eight exons (Ferrara, et al., Endocr. Rev., 13:18-32 (1992); Tischer, et al., J. Biol. Chem., 806:11947-11954 (1991); Ferrara, et al., Trends Cardio Med., 3:244-250 (1993); Polterak, et al., J. Biol. Chem., 272:7151-7158 (1997)). Human VEGF isoforms consists of monomers of 121, 145, 165, 189, and 206 amino acids, each capable of making an active homodimer (Polterak et al., J. Biol. Chem, 272:7151-7158 (1997); Houck, et al., Mol. Endocrinol., 8:1806-1814 (1991)). The VEGF121 and VEGF165 isoforms are the most abundant. VEGF121 is the only VEGF isoforms that does not bind to heparin and is totally secreted into the culture medium. VEGF165 is functionally different than VEGF121 in that it binds to heparin and cell surface heparin sulfate proteoglycans (HSPGs) and is only partially released into the culture medium (Houck. et al., J. Biol. Chem., 247:28031-28037 (1992): Park, et al., Mol. Biol. Chem., 4:1317-1326 (1993)). The remaining isoforms are entirely associated with cell surface and extracellular matrix HSPGs (Houck, et al., J. Biol. Chem., 247:28031-28037 (1992); Park, et al., Mol. Biol. Chem., 4:1317-1326 (1993)).
VEGF receptor tyrosine kinases, KDR/Flk-1 and/or Flt-1, are mostly expressed by EC (Terman, et al., Biochem. Biophys. Res. Commun., 187:1579-1586 (1992); Shibuya, et al., Oncogene, 5:519-524 (1990); De Vries, et al., Science, 265:989-991 (1992); Gitay-Goran, et al., J. Biol. Chem., 287:6003-6096 (1992); Jakeman, et al., J. Clin. Invest., 89:244-253 (1992)). It appears that VEGF activities such as mitogenicity, chemotaxis, and induction of morphological changes are mediated by KDR/Flk-1 but not Flt-1, even though both receptors undergo phosphorylation upon binding of VEGF (Millauer, et al., Cell, 72:835-846 (1993); Waltenberger, et al., J. Biol. Chem., 269:26988-26995 (1994); Seetharam, et al., Oncogene, 10:135-147 (1995); Yoshida, et al., Growth Factors, 7:131-138 (1996)). Recently, Soker et al., identified a new VEGF receptor which is expressed on EC and various tumor-derived cell lines such as breast cancer-derived MDA-MB-231 (231) cells (Soker, et al., J. Biol. Chem., 271:5761-5767 (1996)). This receptor requires the VEGF isoform to contain the portion encoded by exon 7. For example, although both VEGF121 and VEGF165R bind to KDR/Flk-1 and Flt-1, only VEGF165 binds to the new receptor. Thus, this is an isoform-specific receptor and has been named the VEGF165 receptor (VEGF165R). It will also bind the 189 and 206 isoforms. VEGF165R has a molecular mass of approximately 130 kDa, and it binds VEGF165 with a Kd of about 2xc3x9710xe2x88x9210M, compared with approximately 5xc3x9710xe2x88x9212M for KDR/Flk-1. In structure-function analysis, it was shown directly that VEGF165 binds to VEGF165R via its exon 7-encoded domain which is absent in VEGF121 (Soker, et al., J. Biol. Chem., 271:5761-5767 (1996)). However, the function of the receptor was unclear.
Identifying the alterations in gene expression which are associated with malignant tumors, including those involved in tumor progression and angiogenesis, is clearly a prerequisite not only for a full understanding of cancer, but also to develop new rational therapies against cancer.
A further problem arises, in that the genes characteristic of cancerous cells are very often host genes being abnormally expressed. It is quite often the case that a particular protein marker for a given cancer while expressed in high levels in connection with that cancer is also expressed elsewhere throughout the body, albeit at reduced levels.
Prostatic carcinoma is the most prevalent form of cancer in males and the second leading cause of cancer death among older males (Boring, et al., Cancer J. Clinicians, 7-26 (1994)). Clinically, radical prostatectomy offers a patient with locally contained disease an excellent chance for cure. Unfortunately, if diagnosed after metastases are established, prostatic cancer is a fatal disease for which there is no effective treatment to significantly increase survival. Recent advances in prostatic cancer diagnosis has allowed the earlier detection of human prostate cancer by use of the PSA test (Catalona, et al., J. Urol., 151, 1283-1290 (1994)). Unfortunately, this early detection has not been accompanied by an improvement in determining which tumors may progress to the metastatic stage (Cookson, et al., J. Urology 154, 1070-1073 (1995) and Aspinall, et al., J. Urology 154, 622-628 (1995)). Since many individuals having prostate cancer are not adversely effected by the cancer, considerable controversy has arisen as to the use of such tests. Thus, methods for early detection and early appreciation of the potential for or of the severity of the cancer, that can be taken into account in treatment of, for example, metastatic disease, as well as treatment of such diseases are desirable.
We have isolated a cDNA encoding the VEGF165 R gene (SEQ ID NO:1) and have deduced the amino acid sequence of the receptor (SEQ ID NO:2) We have discovered that this novel VEGF receptor is structurally unrelated to Flt-1 or KDR/Flk-1 and is-expressed not only by endothelial cells but by non-endothelial cells, including surprisingly tumor cells.
In ascertaining the function of the VEGF165R we have further discovered that this receptor has been identified as a cell surface mediator of neuronal cell guidance and called neuropilin-1. Kolodkin et al., Cell 90:753-762 (1997). We refer to the receptor as VEGF165R/NP-1.
In addition to the expression cloning of VEGF165R/NP-1 cDNA we isolated another human cDNA clone whose predicted amino acid sequence was 47% homologous to that of VEGF165R/NP-1 and over 90% homologous to rat neuropilin-2 (NP-2) which was recently cloned (Kolodkin, et al., Cell 90, 753-762 (1997)). NP-2 binds members of the collapsin/semaphorin family selectively (Chen, et al., Neuron 19, 547-559 (1997)).
Our results indicate that VEGF165R/NP-1 and NP-2 are expressed by both endothelial and tumor cells. (FIG. 19) We have shown that endothelial cells expressing both KDR and VEGF165R/NP-1 respond with increased chemotaxis towards VEGF165, not VEGF121, when compared to endothelial cells expressing KDR alone. While not wishing to be bound by theory, we believe that VEGF165R/NP-1 functions in endothelial cells to mediate cell motility as a co-receptor for KDR.
We have also shown in the Boyden chamber motility assay that VEGF165 stimulates 231 breast carcinoma cell motility in a dose-response manner (FIG. 15A). VEGF121 had no effect motility of these cells (FIG. 15B). Since tumor cells such as, 231 cells, do not express the VEGF receptors, KDR or Flt-1, while not wishing to be bound by theory, we believe that tumor cells are directly responsive to VEGF165 via VEGF165R/NP-1.
We have also analyzed two variants of Dunning rat prostate carcinoma cells, AT2.1 cells, which are of low motility and low metastatic potential, and AT3.1 cells, which are highly motile and metastatic. Cross-linking and Northern blot analysis show that AT3.1 cells express abundant VEGF165R/NP-1, capable of binding VEGF165, while AT2.1 cells don""t express VEGF165R/NP-1 (FIG. 18). Immunostaining of tumor sections confirmed the expression of VEGF165R/NP-1 in AT3.1, but not AT2.1 tumors (FIG. 17). Additionally, immunostaining showed that in subcutaneous AT3.1 and PC3 tumors, the tumor cells expressing VEGF165R/NP-1 were found preferentially at the invading front of the tumor/dermis boundary (FIG. 17). Furthermore, stable clones of AT2.1 cells overexpressing VEGF165R/NP-1 had enhanced motility in the Boyden chamber assay. These results indicate that VEGF165R/NP-1 expression on tumor cells is associated with the motile, metastatic phenotype.
These results indicate that enhanced transcripts (mRNA) and expression of the VEGF165R/NP-1 and NP-2 receptors have a high correlation to disease state in a number of cancers, such as prostate, hemangioendothelioma and breast, particularly metastatic cancers. Accordingly, assaying for enhanced levels of transcript or gene product can be used in not only a diagnostic manner, but in a prognostic manner for particular cancers. Additionally, by blocking such receptors or inhibiting their occurrence, one can inhibit metastasis.
The present invention provides a method of diagnosing cancer, especially prostate cancer, breast cancer, and hemanyioendothelioma in a patient by measuring levels of VEGF165R/NP-1 or NP-2 in a biological specimen obtained from the patient. Levels of VEGF165R/NP-1 or NP-2 in the sample greater than a base line level for that type of specimen is indicative of cancer. Biological specimens include, for example, blood, tissue, serum, stool, urine, sputum, cerebrospinal fluid and supernatant from cell lysate. The determination of base lines and comparison levels is by standard modes of analysis based upon the present disclosure.
In another aspect, the present invention provides a method of prognosis in an individual having cancer, the method comprising:
a. obtaining a tumor sample from said individual;
b. measuring VEGF165R/NP-1 or NP-2 amounts to obtain an VEGF165R/NP-1 level in said sample;
c. correlating said VEGF165R/NP-1 levels with a baseline level; and correlating levels of VEGF165R/NP-1 or NP-2 higher than the baseline with an indication of unfavorable prognosis and levels of VEGF165R/NP-1 or NP-2 at the baseline or less with a favorable prognosis. VEGF165R/NP-1 mRNA or protein may be measured to obtain VEGF165R/NP-1 levels.
In accordance with the present invention, expression of VEGF165R/NP-1 or NP-2 in a tumor sample greater than a base line level for that particular tissue indicates a higher risk of tumor metastasis.
In yet another aspect, the present invention provides a method for determining the metastatic potential of a tumor by measuring the level of VEGF165R/NP-1 or NP-2 expression in the tumor. Expression of VEGF165R/NP-1 or NP-2 in said tumor greater than a base line level for that particular tissue indicates an increased metastatic potential.
In yet another embodiment, changes in condition can be monitored by comparing changes in VEGF165R/NP-1 or NP-2 expression levels in the tumor in that subject over time.
In the methods of the present invention, levels of VEGF165R/NP-1 or NP-2 can be ascertained by measuring the protein directly or indirectly by measuring transcript (mRNA) encoding VEGF165R/NP-1 or NP-2. mRNA levels can be measured, for example, using an RNA dependent polymerase chain reaction. e.g., reverse transcriptase PCR or Northern blot analysis. DNA chip technology may also be used to measure mRNA levels.
Base line levels can readily be determined by measuring levels of VEGF165R/NP-1 or NP-2.in sample of disease free individuals.
The present invention also provides of a method for measuring VEGF165R/NP-1 or NP-2 levels in non-neuronal tissues which comprises the steps of:
a. contacting a biological specimen with an antibody or antibody fragment which selectively binds VEGF165R/NP-1 or NP-2, and
b. detecting whether said antibody or said antibody fragment is bound by said sample and thereby measuring the levels of VEGF165R/NP-1 or NP-2.
In still another embodiment of this invention, the receptor can serve as a target for compounds that disrupt its function. Such compounds include, for example, VEGF antagonists, compounds that bind to NP-1 or NP-2 and antibodies that specifically binds the receptor at a region that inhibits receptor function. For example, one can add an effective amount of a compound that binds to NP-1 to disrupt receptor and thus inhibit metastasis. In another embodiment, one can use such VEGF165R/NP-1 or NP-2 cells in an assay to discover compounds that bind to or otherwise interact with these receptors in order to discover compounds that can be used to inhibit metastasis.
Other aspects of the invention are disclosed infra.